A manifold-based framework for studying the dynamics of the vaginal microbiome

The vaginal microbiome plays a crucial role in our health. The composition of this community can be classified into five community state types (CSTs), four of which are primarily consisted of Lactobacillus species and considered healthy, while the fifth features non-Lactobacillus populations and signifies a disease state termed Bacterial vaginosis (BV), which is associated with various symptoms and increased susceptibility to diseases. Importantly, however, the exact mechanisms and dynamics underlying BV development are not yet fully understood, including specifically possible routes from a healthy to a BV state. To address this gap, this study set out to characterize the progression from healthy- to BV-associated compositions by analyzing 8026 vaginal samples and using a manifold-detection framework. This approach, inspired by single-cell analysis, aims to identify low-dimensional trajectories in the high-dimensional composition space. It further orders samples along these trajectories and assigns a score (pseudo-time) to each analyzed or new sample based on its proximity to the BV state. Our results reveal distinct routes of progression between healthy and BV states for each CST, with pseudo-time scores correlating with community diversity and quantifying the health state of each sample. Several BV indicators can also be successfully predicted based on pseudo-time scores, and key taxa involved in BV development can be identified using this approach. Taken together, these findings demonstrate how manifold detection can be used to successfully characterize the progression from healthy Lactobacillus-dominant populations to BV and to accurately quantify the health condition of new samples along the route of BV development.


INTRODUCTION
The human body is inhabited by bacterial communities, collectively known as the human microbiome.These communities inhabit multiple sites, including the human gut, skin, and vagina.The composition of the vaginal bacterial population, specifically, can have a major impact on women's health in multiple ways, for example, by interfering with the proliferation of harmful organisms 1,2 .
In an attempt to better understand and characterize the composition of the vaginal microbiome, researchers have sought to cluster the various compositions observed in this community into distinct groups.Such studies have shown that there are at least five major typical compositions of the vaginal microbiota, referred to as community state types (CSTs) 3,4 .Each CST is defined according to the dominant species in the community or the combination of species in this state (see also Table 1).Specifically, several Lactobacillus-dominated compositions exist, including L. crispatus-, L. gasseri-, L. iners-, and L. jensenii-dominated communities, and are referred to as CST I, CST II, CST III, and CST V, respectively.Populations composed of other anaerobes (i.e., non-Lactobacillus-dominated), such as Gardnerella, Prevotella, and Atopobium, are referred to as CST IV.CSTs can be further separated into subgroups (subCSTs).For example, CST IV is further stratified into CST IV-A (dominated by Lachnocurva vaginae; BVAB), CST IV-B (dominated by Gardnerella vaginalis), or CST IV-C (a diverse collection of anaerobes) 5 .Similarly, within the Lactobacillus-dominant CSTs, CST I and III are divided into two subgroups, A and B. Subgroup A is composed of a higher abundance of the focal Lactobacillus species, while the subgroup B is composed of a lower abundance of that species.Finally, CST IV-C is also divided into five groups (labeled 0 to 4), while IV-C0 is a more diverse population and the other groups are dominated by a BV-related bacterium which is not G. vaginalis or BVAB.
Notably, different CSTs are also tightly associated with various physiological and clinical phenotypes.Lactobacillus-dominant CSTs, for example, are generally characterized by low levels of pro-inflammatory cytokines and low pH, which is ascribed to the production of lactic acid by the dominating Lactobacillus species 3,6 .Non-Lactobacillus CSTs, in contrast, can be accompanied by high pH and unpleasant symptoms, such as mal-odor and abnormal discharge.CST IV is often referred to as Bacterial Vaginosis (BV), a common dysbiotic condition.BV is also associated with multiple adverse gynecologic consequences, including an increased risk of preterm birth 7 , pelvic inflammatory disease 8 , acquisition of STIs including HIV 9 , and human papillomavirus 10 .Unfortunately, even though BV is very common, its etiology and development are still not clear, calling for a more principled mapping of its dynamics.
Indeed, the composition of the vaginal microbiome is not static, and changes throughout a woman's life, from childhood to reproductive age, and during pregnancy and menopause 11 .These changes are ascribed to resource availability in the vaginal environment, such as the abundance of glycogen in the vaginal epithelium, which in turn seems to be highly affected by estrogen levels 12 .Changes may also occur at much shorter timescales, for example, in correspondence to the menstrual cycle 13 .Sharp compositional shifts can also be observed, for example, following sexual intercourse and other host habits [13][14][15][16] .These various processes and factors are clearly not independent, making the dynamics of the vaginal microbiome challenging to comprehensively characterize, in spite of the relatively simple composition of that microbiome.
Clearly, while changes in the composition of the microbiome occur in a high-dimensional space (i.e., spanning all possible community compositions), microbial dynamical processes (such as the transition from a healthy state to BV) may potentially follow relatively common patterns, and accordingly, likely progress along a low-dimensional trajectory embedded in this high-dimensional space.The detection and characterization of such trajectories, however, is a challenging task as the vaginal microbiome is affected and may be perturbed by many factors.Indeed, several approaches were used to describe the dynamics of the vaginal microbiome.Gajer et al., for example, have used an ordination analysis to show a low dimension representation of vaginal microbiome compositions (based on cross-sectional and longitudinal data) and the progression of each woman's microbiome over time 3,13 .This study clearly illustrated the continuous nature of transitions between CSTs, albeit, without a qualitative interpretation of intermediate stages.Other studies used integration of time-series data across individuals by alignment of longitudinal vaginal samples 17,18 .The aligned data in these studies revealed some interesting insights, including, for example, an antagonistic behavior between L. iners and Atopobium.An additional longitudinal metagenomic study of healthy women with high risk for BV proposed that BV formation is initiated by early colonizers (such as Gardnerella vaginalis), creating a more favorable environment for other BV-related bacteria 19 .However, to date, a rigorous approach for describing the route of transition between Lactobacillus-and non-Lactobacillus-dominant CSTs has not yet been presented, calling for a robust framework that enables identification of complex dynamical processes in this high-dimensional data.
Interestingly, dynamical processes and progression trajectories can be potentially identified not only via longitudinal data analysis, but also via the analysis of massive cross-sectional datasets.Such datasets can be viewed as a collection of snapshots into the underlying dynamical process, such that each sample provides information about a specific point along the progression trajectory.Identifying low-dimensional trajectories embedded in this high-dimensional space and ordering samples along the identified trajectories can therefore both help characterizing the dynamical processes involved and enable labeling samples according to their location along such processes.An example of this approach has been presented in a study by Li et al., who suggested a model that recapitulated longitudinal progression of the gut microbiome in Crohn's disease 20 .Based on a combination of clustered samples and a principal tree (which represents the topology of trajectories and alterations in the microbial composition), they proposed a double bifurcating model of microbial alterations that occur during Crohn's development.While this model successfully captures the complex structure of disease progression, the low sample size limits the model's robustness and applicability.Another study applied the pseudo-time approach to examine the structure of the gut microbiome throughout the human lifespan 21 .This research discovered distinct clusters within the microbial structure that were linked to the proportions of specific bacterial genera, along with associations between several functional characteristics and the endpoints of these clusters.While yielding intriguing findings, pseudo-time analysis in this study was not designed to be associated with a disease state, but rather to examine microbiome alteration along various branches in the low-dimensional space.Similar analyses of dynamical processes in complex biological systems are also common in single-cell studies, where high dimensionality and high resolution data provide snapshots of single-cell states along some processes, such as cell maturation or differentiation 22,23 .Such analyses then aim to identify the low-dimensional trajectory (or "manifold") embedded in the high-dimensional space of cell states and label each cell according to its location along this trajectory.
In this study, we similarly use a framework inspired by singlecell analysis approaches for manifold detection to identify and characterize the dynamics of the vaginal microbiome.Specifically, we aim to characterize the trajectories of progression from Lactobacillus-dominant populations to a non-Lactobacillus population.Identifying low-dimensional trajectories in the vaginal microbiome composition space and projecting new crosssectional or longitudinal samples on these trajectories allow us to both map the potential routes of the vaginal microbiome and to trace the dynamics along these trajectories (e.g., by examining the progression of a certain woman along these trajectories over time).Moreover, combining cross-sectional and longitudinal data allows us to quantitatively characterize the dynamics of various women, and to compare, for example, the progression along various trajectories to multiple clinical variables.Combined, our findings provide a more principled perspective for examining the vaginal microbiome dynamics, including complex transitions between CSTs and BV development.

RESULTS
A manifold detection approach for characterizing the dynamics of the vaginal microbiome Aiming to comprehensively characterize the dynamics of BV development, we utilized a computational framework that can both identify various possible low-dimensional routes in the microbiome compositional space, and place each sample along these routes.This framework first takes as input a large collection of microbiome samples (from either cross-sectional or longitudinal studies; Fig. 1a) and utilizes a previously introduced manifold detection algorithm 24 to reduce the dimensionality of the data and to identify a low-dimensional manifold that describes the distribution of all input samples in this space (Fig. 1b).This algorithm further labels each sample according to its location on the manifold and the distance of this location to a pre-defined set of root samples (Fig. 1c).This label, referred to in the single-cell literature as pseudo-time, quantitatively assess how close, along the manifold's available routes, is the sample of interest from some given state.In our analysis, we used highly diverse BV samples (mostly assigned with CST IV-B) as root and defined Table 1.Dominant taxa and key characteristics in main CSTs (as previously described 5,38  pseudo-time between 0 and 1, such that 0 denotes a sample far from the BV state (the "healthiest" composition) and 1 as the root (a BV composition).Specifically, in this study, we used the Partition-based graph abstraction (PAGA) manifold detection algorithm 24 , which projects the samples on a low-dimensional space and then utilizes the distance matrix based on the lowdimensional graph to produce pseudo-time labels (see "Methods" for full details).
In addition, we also use a previously developed method for determining the CST of each sample 5 .Since samples from a given CST are also generally more similar to one another than to samples from a different CST, samples from each CST tend to form a distinct route (or an "arm") in the above identified manifold (Fig. 1d).Finally, given a new set of samples (i.e., that were not part of the input and the detected manifold, such as samples from some women in a new longitudinal study), we match each new sample to its nearest neighbor sample on the manifold, using the location and pseudo-time label of this nearest neighbor to label the new sample (Fig. 1e).
Combined, this procedure assigns each sample a quantitative measure of its progression along possible routes from healthy to BV states, allowing us to study the dynamics of the vaginal microbiome.For example, focusing on longitudinal samples from a single woman, and comparing the pseudo-time label of these samples to the chronological time in which they were obtained, allow us to comprehensively map each woman's trajectory along the route of BV development (Fig. 1f).In addition, we can compare samples' pseudo-time labels to various clinical parameters, to examine correspondence with BV progression (Fig. 1g), or use pseudo-time to predict clinical BV-related variables.

Analysis of vaginal community dynamics in a large-scale dataset collection via manifold detection framework
To assess the ability of the framework described above to identify BV developmental trajectories in the vaginal microbiome, we analyzed five different datasets, including three cross-sectional datasets and two longitudinal datasets (Table 2).Briefly, the crosssectional datasets, combined, include thousands of samples, mostly from reproductive-age asymptomatic women.The longitudinal datasets, include again thousands of samples combined, with one dataset of 83 women sampled daily (with ~70 samples per woman), and another of 84 women sampled bi-monthly (with 2-7 samples per woman).Samples with insufficient coverage were filtered and discarded from downstream analyses.In total, our dataset collection after filtration included 8541 samples.Available metadata varied across datasets, with some datasets providing detailed data about age, ethnicity, and various clinical BV indicators and criteria, while others included more limited metadata.Complete information about these datasets and available pertaining information can be found in the Methods.
The CST of each sample in our dataset collection was determined using the VALENCIA algorithm 5 .Across all five datasets, 57% of the samples were assigned to Lactobacillusdominant CST, in general agreement with other studies 3,25 (Supplementary Fig. 1a).CST assignment also produced a similarity score for each sample, indicating how much the sample resembled the core CST as defined by VALENCIA 5 (see "Methods").The mean similarity score across all samples was 0.75, and varied between CSTs, with a mean similarity of 0.86 in the main Lactobacillus CSTs (I-A, II, III-A and V; Supplementary Fig. 1a), 0.79 in the secondary Lactobacillus CSTs (I-B and III-B), and 0.56 in non-Lactobacillus CSTs (IV-A, IV-B and IV-C), again in accordance with similarity scores reported by other studies 26 .We also calculated the Shannon-diversity index for each sample.As expected, Shannon-diversity index varied substantially between communities, with CST IV-C0 and IV-A exhibiting the highest mean diversity (2.75 and 2.24, respectively), and CST I-A and III-A the lowest diversity (0.29 and 0.47, respectively; Supplementary Fig. 1b).These values match variation in diversity across CSTs as previously reported 5 .Additionally, to allow us to evaluate the capacity of our framework to assign meaningful pseudo-time labels to "new" samples, we held out a set of samples from our dataset, using only the remaining samples for identifying and characterizing the manifold as described above.Specifically, we randomly selected 30 women from one of the cross-sectional datasets (AVPVC) 3 and 12 women from one of the longitudinal datasets (UAB 27 ; each with multiple daily samples), for a total of 515 samples.These held-out samples were not included in our pseudo-time analysis, and instead were each mapped to its nearest neighbor (using the Bray-Curtis distance metric) on the manifold and assigned the pseudotime and CST of that nearest neighbor.
Having constructed and processed this dataset collection, we finally applied the framework described above to samples from the five datasets.Dimensionality reduction, as a part of the PAGA package, resulted in a graph with distinct "arms", each could be assigned to a specific CST (Fig. 2a, inset).The arms of CST-I and III were the longest, and each could be further partitioned into the main subCST, A (with sample located closer to the periphery of the graph), and the secondary subCST, B (with samples closer to the center of the graph and to BV state samples).This illustrates the gradual changes in vaginal community along routes of BV development, as well as the prevalence of these CSTs 3,5 .Notably, samples from the five different datasets were relatively wellspread across the UMAP space (Fig. 2b), confirming that the result manifold is not an artifact of some study-specific bias.This manifold was then used to calculate the pseudo-time of each sample, based on provided root samples (Fig. 2c; see "Methods").In our analysis, we defined samples with positive Amsel's test (i.e., three positive criteria out of four) and with Shannon-diversity index >3.5 (considered less healthy), as root samples.

Pseudo-time labels' association with BV-related clinical indicators
One of the potential advantages and application of our pseudotime approach is that it provides an objective quantification of a sample's progression toward a BV state, based solely on observed trajectories along the vaginal microbiome compositional space.Indeed, for example, we found that the mean pseudo-time of Lactobacillus-dominant CSTs was 0.72, whereas that of non-Lactobacillus CSTs was 0.91, indicating that Lactobacillus populations are further away from an unhealthy state, as expected.As stated above, in our work, lower pseudo-time signifies greater distance from the BV state, and hence a lower mean pseudo-time of samples from a particular CST suggests that these samples are on average closer to the healthy state.To further confirm the utility of the obtained pseudo-time label as markers for BV progression, we next examined how they correlate with various ecological and BV-related features, such as Shannon-diversity index, Nugent score, and Amsel's criteria.We first focused on community diversity, acknowledging that a diverse vaginal population is generally considered less healthy and closer to BV state.This analysis was conducted separately for each arm on the manifold (based on Fig. 2a), since BV developmental patterns may vary across different source CSTs.As expected, we found that Shannon-diversity index was highly correlated with pseudo-time across all arms, with R 2 of 0.79, 0.66, 0.8 and 0.69 in arms of CST I, II, III and V, respectively (Fig. 3a).To evaluate whether the correlation between Shannon-index with pseudo-time is influenced by potential dataset effects, we also applied a random effects model, using the dataset identity as a random effect, and found that this correlation was still significant (p < 0.0001).As an additional evaluation, we also confirmed that the correlation between Shannon-diversity and pseudo-time remains significant when calculated for each dataset separately (Supplementary Table 1).Moreover, held-out samples (see above) that were assigned with pseudo-time labels according to their nearest neighbors' labels have also exhibited a high correlation between their Shannon-diversity values and the assigned pseudo-time label in the healthy available arms (CST I and III; Supplementary Fig. 2).Since root samples in our manifold were also defined as those with the highest Shannon-diversity index (along with positive Amsel's test result), and other CSTs are generally defined by the dominance of one phylotype (and are hence less diverse), these correlations are not necessarily surprising, yet, the strong correlation suggests a good fit of the samples' placement along the manifold and their bacterial population's composition.Importantly, however, examining the relationship between pseudo-time and clinical BV indicators, we again found similar, albeit somewhat weaker correlations.Specifically, Nugent score in all arms, except for IV-C, exhibited a significant correlation with pseudo-time values (Spearman correlation; FDR < 0.05).Furthermore, pseudo-time labels significantly differed between samples with positive and negative Amsel's test results in arms I, III, IV-A, and IV-B.Arms II and V were excluded from this analysis due to insufficient sample size, while arm IV-C showed no significant difference (Supplementary Table 2).As for Amsel's specific criteria, we found that pseudo-time showed significant correlations with pH in three arms (III, IV-B, and IV-C) and significant differences Given our primary objective, which is to elucidate the development of BV through pseudo-time analysis, we next aimed to examine how well BV-indicators can be predicted using pseudo-time labels.To this end, we calculated for each BVindicator a Receiver Operating Characteristic (ROC) curve, describing how well can the pseudo-time label separate between positive and negative values of this indicator given different pseudo-time thresholds (Fig. 3b).Then, the area under the ROC curve (AUC) was calculated to quantify the overall predictive accuracy.To further assess the information captured in the pseudo-time labels, we compared the obtained ROC AUC to predictions using shuffled labels.Notably, samples defined as root samples for the manifold detection analysis were discarded from this analysis.Here, we focused on six BV indicators: high Nugent score, positive Amsel's test, and the four criteria of Amsel (elevated pH, positive whiff presence of clue cells, and abnormal vaginal fluid).Since not all indicators were available in all datasets, we limited the analysis of each indicator to the dataset in which it was provided.Specifically, Nugent score was available only in the UAB study, Amsel's test and pH were available in the PHS, UAB, and PVI studies, and the remaining Amsel's criteria were available only in the PHS and PVI datasets.Evidently, our analysis revealed that pseudo-time could effectively predict all BV indicators, with ROC AUC ranging between 0.65 (and for all indicators except vaginal fluid, 0.72) to 0.88.The relatively low predictability of vaginal fluid, may be attributed to the dispersed distribution of samples with abnormal fluid on the manifold, and in agreement with one dataset from the original PVI study, where only BVAB1 was associated with abnormal vaginal fluid, compared to more than ten taxa significantly associated with other Amsel's criteria 28 .Repeating this analysis using only the held-out samples, we observed similar predictability (since held-out samples were chosen from the AVPVC and UAB datasets, only the high Nugent score, positive Amsel's test, and elevated pH indicators were available, with only 7 samples positive for Amsel's test).

Pseudo-time labels' association with menstrual cycle and BV-related metabolites
Given the significant influence of the menstrual cycle on fluctuations in vaginal population composition 13,29 , we next aimed to investigate the relationship between pseudo-time labels and menstruation.We hypothesized that pseudo-time would exhibit larger fluctuation toward unhealthy state (i.e., higher pseudo-time) prior to and during menstruation in comparison to fluctuations in other times.To investigate this, we calculated the difference in pseudo-time between each two consecutive samples, and compared the observed differences in the period ranging from 2 days before menstruation to the end of menstruation with differences observed in all other times.Our analysis indeed demonstrated a statistically significant increase in fluctuation toward higher pseudo-time in menstruation compared to non-menstruation periods.This difference was observed among the samples used in our manifold detection analysis (Wilcoxon test, Supplementary Fig. 3), with a similar trend observed in the held-out samples (but did not reach statistical significance).
In order to validate the effectiveness of our pseudo-time framework, we next examined the relationship between pseudotime labels and BV-associated metabolites, such as biogenic amines [30][31][32][33] .These amines include, for example, the metabolites cadaverine, putrescine, and tyramine, which were previously linked to increased likelihood of transitioning from Lactobacillusdominant CST to non-Lactobacillus CST.Indeed, data about the level of five biogenic amines (cadaverine, putrescine, spermine, spermidine, and tyramine), was available for samples from the UAB dataset used in our pseudo-time analysis 31 .We found the levels of cadaverine, putrescine, and tyramine (but not spermidine and spermine) were significantly higher in samples labeled with high pseudo-time (>0.9), compared to samples with low pseudotime (p = 0.00001, 0.00029, 0.0044, respectively; Wilcoxon test; Supplementary Fig. 4), in agreement with the association between BV and these three biogenic amines reported above [30][31][32][33] .Combined, these analyses revealed robust correlations between pseudo-time labels and multiple key indicators of BV and BVrelated metabolites.
Individual trajectories of women from the longitudinal dataset along the manifold Since our dataset collection includes longitudinal samples, wherein multiple samples were obtained from some women over time, we compared for each such woman the calculated samples' pseudo-times with the chronological time at which the samples were obtained, thereby characterizing the specific trajectory of each woman along the manifold.Importantly, our pseudo-time calculation did not utilize information about which sample originated from which woman or at what chronological time in any way.As evident from the top plots in Fig. 4, observed trajectories varied substantially between women.For example, woman UAB103 (Fig. 4a) demonstrated a relatively stable trajectory, remaining on the same healthy arm of the manifold (CST I) throughout the experiment's duration, although moving back and forth (i.e., closer and further from the BV state) along this arm.In contrast, woman UAB059 (Fig. 4b) exhibited a recurrent transition from a healthy state to a BV state (generally in sync with the menstrual cycle), moving from one arm to another.Finally, woman UAB002 (Fig. 4c) demonstrated frequent sharp transitions, moving between multiple arms on the manifold (including three different healthy arms and two BV-associated arms) without any discernible pattern.The trajectories of all women from this dataset on the manifold can be found in Supplementary Fig. 5, and pseudo-time progression along chronological time can be found in Supplementary Fig. 6.These observed differences in women's trajectories may reflect various factors that affect community behavior.For example, a continuous increment in pseudo-time along chronological time (as seen, for example, in subjects UAB001; Supplementary Fig. 6) may represent a gradual progression toward BV, whereas, a sharp increase in pseudo-time (as seen in subject UAB075) might imply a specific event or exogenous perturbation that caused this shift.One such perturbation, for example, may be the use of medications for treating BV, aiming to shift the microbial population toward a healthier state.Although the efficacy of such medications can vary among women 34,35 , we hypothesized that the use of such medications should lead to a decrease in pseudotime (i.e., a movement along the manifold toward a healthier state).To test this hypothesis and investigate the impact of BV medication on pseudo-time, we used available data from the UAB dataset, and analyzed samples collected during medication use, as well as 1 day before and after treatment.Among the ten instances of BV medication usage across 13 incidents of 11 women, nine indeed showed a consistent decrease in pseudo-time during this period (Supplementary Fig. 7), highlighting the potential of our pseudo-time labels as an effective model of temporal microbiome dynamics.

Shifts in taxa abundances during BV development
Even though BV is a highly common disease worldwide, its etiologic agent is yet unknown.Specifically, our understanding of disease development in women with recurrent BV or with slow progression toward disease (i.e., in women not affected by sexual intercourse or another unique event 36 ), is lacking.The vaginal microbiome composition manifold, presented in Fig. 2, can serve as a comprehensive map of the potential routes of the vaginal microbiome, allowing us to quantify changes in bacterial abundances along various manifold's arms, each representing a different route of BV development.For this purpose, samples were separated again into the different arms and ordered by pseudotime label.To address the noisy and stochastic nature of the data, we further used a sliding window and calculated the average relative abundance of each taxon along pseudo-time (see "Methods"; Fig. 5).An increase in a taxon's abundance with pseudo-time values implies a potentially important role in BV development, especially if this increase is observed in multiple arms.For example, G. vaginalis, Finegoldia, and Streptococcus increased in abundance with pseudo-time in all arms.G. vaginalis, specifically, increased in abundance substantially with pseudotime, reaching a relative abundance of 2.3-11% in the samples with the highest pseudo-time, although these samples are still classified as being in a healthy CST.This finding supports the association between G. vaginalis BV that was previously shown 37 , and suggests it has an important role in BV development from all healthy CSTs.Interestingly, all Lactobacillus species also tended to increase in abundance in the arms in which they were not the dominant species, potentially owing to a general increase in diversity and a corresponding shift from a single Lactobacillus species-dominant community to a more diverse community.Lastly, the abundance of several taxa increased in specific arms, such as Corynebacterium (arms I, II and V), Bifidobacterium (II and V), Anaerococcus (I and II), Prevotella (II), and Megasphaera (III).Even though these taxa's abundances were relatively low throughout the trajectory, this might suggest different mechanisms of BV progression (that are driven by different bacterial species) from different healthy CSTs.

DISCUSSION
With the goal of comprehensively characterizing the dynamics of the vaginal microbial population, we applied a framework of manifold detection inspired by common single-cell analysis.The detected manifold can serve as a map of potential routes from healthy CSTs to BV.Moreover, this approach allows us to assign each sample with a pseudo-time label, describing the location of each sample on the manifold in relation to healthy vs. BV states, hence offering a proxy for the status of the sample.Our findings provide insights into the structure of vaginal microbiome dynamics and shed light on common vaginal trajectories and BV etiology, while offering an ability to evaluate the state of new samples.
Our analysis of the obtained pseudo-time labels revealed a robust association between these labels and multiple BV-related clinical parameters.With that in mind, it is worth noting that pseudo-time labels are obtained based on the bacterial composition alone, suggesting that our framework can be used to provide complementary and valuable insights into the health status of a given sample, and to infer the condition of a sample with respect to BV development stage.
Notably, amongst the BV indicators we examined, abnormal vaginal fluid exhibited only limited association with pseudo-time.Indeed, as shown in the original PVI study 28 , only BVAB1 was associated with abnormal vaginal fluid in the PVI dataset, in contrast to the PHS dataset, suggesting that this symptom needs to be further explored in different contexts.Specifically, the PVI dataset was obtained mostly from women of African ethnicity, and given the observed variation in the vaginal microbiome across ethnicities 3 , a larger dataset of more diverse ethnicities may be required.Unfortunately, the majority of samples in our study lacked accessible ethnicity data or reported ethnicity using different labeling systems, impeding our assessment of the impact of ethnicity in relation to pseudo-time.
Our analysis of microbial compositions along the vaginal microbiome manifold further revealed bacterial taxa whose abundances change in association with pseudo-time in the different manifold arms, potentially highlighting the role of various microbes in BV development.Interestingly, while the abundances of G. vaginalis, Finegoldia, and Streptococcus increased with pseudo-time in all arms, shifts in the abundances of other taxa arm-specific, suggesting different mechanisms of transition from the distinct Lactobacillus CSTs toward BV.G. vaginalis was considered associated with BV, however, its association with BV Fig. 4 A manifold-based characterization of the trajectories of three representative women from the daily longitudinal dataset (UAB).The top plot in each panel (a-c) illustrates the trajectory of the woman on the detected manifold.Gray dots represent the UMAP visualization of the vaginal microbiome composition manifold, and colored dots represent the locations of samples obtained from a specific woman, where each color represent the determined subCST of the sample.Black arrows denote the direction of the woman's trajectory, showing the order of the samples based on the time they were obtained in the experiment.The bottom plot in each panel illustrates pseudo-time progression of the woman, as a function of chronological time.Dots colors are as in the top plots.Red triangles at the bottom of the plot represent selfreported menstruation, where size indicates menstruation spotting (small), medium or heavy bleeding (large).
was shown to be strain-dependent 38,39 , therefore, further analysis, for example using shotgun sequencing, is required to evaluate its contribution to development.Finegoldia and Streptococcus were also previously identified as BV-related bacteria, together with many of the arm-specific taxa we report in our analysis 27,28,40 , yet, the relation of the arm-specific taxa to BV development from a specific CST source needs to be further evaluated.Notably, Prevotella-a taxon that has been hypothesized to have a dominant role in BV development (e.g., by creating a biofilm with G. vaginalis 19,41 )-appeared in our analysis only in one Lactobacillus-dominant arm (II), calling for further investigation into its role in BV development from other healthy CSTs.Other species that increased with pseudo-time might grow with G. vaginalis to form a BV biofilm, including Finegoldia 37 , Streptococcus 42 and Atopobium 43 .These observations suggest that examining the progression of BV from the perspective of the previous healthy state is essential for gaining insights into the interactions between bacterial taxa and the changes in their abundance in the formation of BV biofilm.
While the observed associations between pseudo-time labels and various clinical parameters attest to the validity of our approach, it is important to acknowledge the limitations of the manifold framework.Specifically, it is worth noting that pseudotime labels are ultimately based on changes in the bacterial composition of the microbiome, and hence correlations between pseudo-time and measures closely related to the bacterial population (such as Shannon-diversity or Nugent score) are perhaps not surprising.As such, our framework and the obtained pseudo-time labels can be viewed as a powerful approach to summarize the complex and highly dimensional microbiome data, into a meaningful, clinically-relevant metric, offering a spatial perspective (i.e., manifold arm and location along the arm) for these data.Another limitation in our framework is the need to define root samples for the pseudo-time analysis.For our analyses, we used Amsel's positive samples with high diversity as root samples, however, the ambiguous definition of BV, along with missing information in our metadata, make it difficult to determine the ideal root samples that should be used to represent BV.Additionally, even though the datasets utilized in this study provide a massive number of samples, they may still not describe the full range of variation that may be present in the vaginal human population.A larger and more diverse dataset may thus be necessary to characterize the vaginal microbiome manifold more precisely.Lastly, an important issue to consider in assessing the utility of manifold detection approaches for studying the vaginal microbiome is the marked differences between the properties of microbiome data and that of single-cell transcriptome data, to which manifold detection approaches are generally applied.Specifically, while single-cell data often describe a pre-defined, well-characterized process (such as cell differentiation), the dynamics of microbial populations are generally more noisy, including abrupt changes, exogenous perturbations, and individual-specific variation.Furthermore, microbial processes are generally not strictly regulated and not necessarily directional, and hence might exhibit back and forth transitions between different states.However, despite these differences, our study suggests that a manifold detection framework can still be applied to vaginal microbiome data and to reveal valuable insights into important microbial processes.
Combined, our analyses and findings above provide a proof of concept for the use of manifold detection methods to map vaginal bacterial samples onto a low-dimensional space, enabling the characterization of potential routes from healthy vaginal states to BV and offering a deeper understanding of vaginal microbiome dynamics.Moreover, the pseudo-time labels obtained using this approach can serve as a powerful indicator of vaginal microbiome status, and enhance our ability to assess a woman's health, potentially facilitating early diagnosis and treatment.

Datasets and data processing
Five datasets were analyzed, three cross-sectional and two longitudinal (see also Table 2).The first cross-sectional dataset, termed VCU (Bioproject PRJNA46877), is a part of the VaHMP study 44 , which includes 3956 samples.The data was collected from women of reproductive age, and the metadata was not published.The second dataset 3 , termed AVPVC (Bioproject SRA022855), is composed of 396 women of reproductive age, and includes information about Nugent score, pH, age, and ethnicity.The third cross-sectional study, termed PHS 45 (Bioproject SRA051298), includes 242 women and information regarding Amsel's criteria such as pH, clue cells, vaginal fluid and whiff test.The first longitudinal dataset 27 , termed UAB (Bioproject PRJNA208535), included 3700 samples and was partitioned into two subsets for processing, due to differences in the sequencing platform and sequenced region.The first subset includes 1658 samples sequenced with 454 pyrosequencing of the V1-V3 region.It was collected from 25 women of reproductive age every day for 70 days, and contains data regarding menstruation, age, ethnicity, Nugent score, pH, as well as additional important BVrelated information such as BV medication, Amsel's test results, and specific BV self-reported symptoms.The second subset includes 2042 samples from 176 women of the V3-V4 region, sequenced via Illumina.It contains data regarding menstruation, Nugent score, and pH, however, does not include BV-related information.The second longitudinal dataset, termed PVI, includes 497 samples from 84 women (Bioproject PRJNA638104) 28 .Samples were collected from four clinics, three clinics in Kenya and one in Alabama.Samples were collected once in 2 months, Fig. 5 Bacterial taxa's relative abundances as a function of by pseudo-time.Each plot represents the taxa's abundances in each healthy manifold arm (I, II, III, and V).Taxa's abundances were averaged using a sliding window approach to reduce noise.
with 2-7 samples per woman.This dataset includes information about specific Amsel's criteria state, such as pH, clue cells and vaginal discharge.
Data were acquired from NCBI as files and were processed via DADA2 46 , resulting in amplicon sequence variants (ASVs) at high resolution.All datasets were filtered for more than 1000 reads in each sample and more the 100 reads for each ASV, to exclude very rare ASVs in each dataset.The filtration step resulted in 3879 samples in the VCU dataset, 385 samples in the AVPVC dataset, 185 of the PHS dataset, 3597 samples in the UAB dataset and 495 samples from the PVI dataset.In total, 8541 samples were processed.Taxonomic classification was assigned to each ASV using the RDP Naïve Bayesian Classifier 47 trained with the SILVA 16S rRNA gene database (version 138.1) 48.Further important species classification (Lactobacillus, Gardnerella, Prevotella, Atopobium, Shuttleworthia and Sneathia) was done with speciateIT (https://github.com/Ravel-Laboratory/speciateIT).In the second cross-sectional dataset and the subset sequenced with Illumina, ASVs assigned to L. ultunensis were changed to L. crispatus, due to their observed part in the population compared to the results.Then, samples were assigned to a specific community state type (CST) using the VALENCIA algorithm 5 .VALENCIA implements a distance-based metric to classify each sample, based on the similarity score to the centroid of CSTs identified in a reference set.Notably, the VALENCIA method is well-established and enjoys widespread adoption in the vaginal microbiome research community.It specifically minimizes potential dataset variability by utilizing pre-defined centroids that are based on a large-set of vaginal microbiome samples, in contrast to methods such as Dirichlet mixture models (DMM) 49 , which may form new clusters based on community composition.All taxonomic units were transformed to relative abundances in each sample.Rare species were removed based on their prevalence (ratio of non-zero samples to total number of samples >0.0001) and their average abundance (>0.00005) across all samples, resulting in 373 taxa in the final analysis.
A framework for vaginal microbiome manifold detection and pseudo-time labeling Aiming to map the potential routes of the vaginal microbiome dynamics, we applied the PAGA 24 algorithm, which is a common approach in the single-cell RNA-sequencing field 50 .The first step of the PAGA algorithm includes dimensionality reduction using PCA.Then, PCA values are projected onto a lower dimensional space (i.e., a manifold) using UMAP 51 (Uniform Manifold Approximation and Projection).UMAP is the default method for dimensionality reduction used in PAGA, and was therefore applied in our analysis rather than other available methods, such as PHATE 52 .Finally, the samples are ordered along the manifold and a pseudo-time label is assigned to each sample using an extension of diffusion maps 53 .Notably, the diffusion-map algorithm requires a pre-defined group of cells/samples denoted as root samples, which represent the beginning of the manifold.In our analysis, we defined root samples as samples with positive Amsel's test and high Shannon-diversity (>3.5).A positive Amsel's test is the best clinical evaluation for BV and high diversity of the microbiome is also considered as a sign for an unhealthy state 5,38 .Pseudo-time is computed by defining coordinates as dominant eigenvectors of a transition matrix that describes random walks between data points, resulting in a quantitative measure of progress through the biological process.In our analysis, the vaginal microbiome composition manifold was detected using 8026 samples of the five datasets mentioned.A total of 515 samples were held out from the manifold analysis, with 485 samples selected at random from the UAB longitudinal dataset representing 12 women, and 30 women selected at random from the AVPVC dataset with one sample per woman.These samples were subsequently utilized to evaluate the projection of new samples onto the manifold as described below.To assign held-out samples with a location on the manifold and a pseudo-time label, samples were mapped onto the manifold using a nearest-neighbor approach, using Bray-Curtis dissimilarity metric 54 based on the microbial composition of the samples.Then, pseudo-time and UMAP values of the nearest neighbor were assigned to the held-out sample for further analysis.

Evaluation of the association between pseudo-time labels and BV indicators
To enhance the assessment of the predictive capacity of pseudotime labels, we focused on appraising the predictive abilities of pseudo-time in conjunction with the Nugent score, Amsel's test result, and the specific Amsel's criteria (i.e., pH, whiff test, occurrence of clue cells, and abnormal vaginal discharge).For this purpose, all BV indicators (including continuous indicators) were categorized into pre-defined value options.A positive Nugent score was defined as Nugent >7, an elevated pH value was defined as pH >5.5, and a positive Amsel's test result was defined as positive symptomatic and asymptomatic Amsel's test outcomes.The utilization of Nugent score and pH values in previous studies has established their relevance in describing the vaginal microbiome state, particularly in relation to BV 5 .The remaining Amsel's criteria derived from the original analysis of the PVI and PHS datasets, including identifying the presence of clue cells at a rate exceeding 20% as a positive test, and categorizing thin, gray, and homogeneous vaginal fluid as indicative of abnormality.Worth to notice that BV indicators were available in different datasets, therefore Nugent score was held from the UAB study alone, pH and Amsel's test from UAB, PVI, and PHS, and all other Amsel's criteria (whiff test, clue cells and vaginal fluid) were obtained from both PVI and PHS studies.To evaluate the prediction of BV indicators using pseudo-time, we employed a receiver operating characteristic (ROC) curve analysis.The ROC curve allowed us to assess the performance of pseudo-time in distinguishing between positive and negative values of each BV indicator.The area under the ROC curve (AUC) was then calculated to quantify the overall predictive accuracy of pseudo-time for BV indicator value classification.In addition, we shuffled indicator's values 100 times to generate ROC curves and AUC calculations based on the shuffled data, to demonstrate the predictive capability of pseudo-time in comparison to a random null.This analysis provided valuable insights into the effectiveness of pseudo-time in predicting BV indicators, aiding in the assessment of its diagnostic potential for BV.

Assessing the interaction between pseudo-time labels and menstrual cycle and biogenic amines
To investigate the association between menstruation and variations in pseudo-time, we computed a pseudo-time differentiation value for each sample, representing the difference in pseudo-time between the current sample and the earlier time-point within the same woman.These pseudo-time differentiation values were categorized into two groups: (1) samples from the previous 2 days of and during the menstruation period, and (2) all other samples.The difference between the pseudo-time differentiation values of these two groups was evaluated using Wilcoxon test in R.
We also evaluated association of five biogenic amines (cadaverine, putrescine, spermidine, spermine, and tyramine), obtained from another study based on samples utilized in the manifold detection framework 31 .These biogenic amines were measured in 79 samples of 19 women, using targeted liquid chromatography-mass spectrometry (LC-MS).Due to the uneven distribution of the samples between CSTs, we have decided to divide pseudo-time into two categories for this analysis.High pseudo-time was defined as pseudo-time >0.9.Biogenic amines

Fig. 1
Fig.1Analysis scheme of the manifold detection framework.a The framework takes as input a high-dimensional microbial data table, describing microbial features of each sample.b Data are then projected onto a lower dimensional space, resulting in a compositional manifold.c Each sample is labeled with a pseudo-time score, calculated based on its distance along the manifold from a predefined group of root samples.d The manifold is also partitioned into "arms" (which represent distinct routes on the manifold and different possible routes to BV), based on the assigning CSTs to the various sample.e New samples are matched to their nearest neighbor on the manifold, obtaining their pseudo-time label and location on the manifold from their nearest neighbor.f Samples from a longitudinal dataset can be mapped to this manifold and used to characterize the trajectory of each woman along the manifold, comparing samples' pseudo-time labels to the chronological time in which the sample was obtained.g Pseudo-time labels associations with various clinical BV-related variables, including community diversity, Nugent score, and pH, can be examined.

Fig. 2
Fig. 2 Visualization of the vaginal microbiome composition manifold and its relation to various sample properties.Each point represents a single sample (from the pooled set of 8026 samples from the five datasets analyzed), where its location was determined by low dimensionality reduction using the PAGA algorithm.a Colors indicate the subCST determined for each sample based on VALENCIA.The inset on the bottom left corner represents the partition of the samples into the different manifold's arms, where each arm is associated with a different CST.b Colors indicate the dataset each sample was obtained from.The UAB dataset was further partitioned according to the sequencing method that was used.c Colors indicate the pseudo-time label that was assigned to each sample based on results from the PAGA algorithm.Black points denote root samples, defined as highly diverse samples (Shannon diversity index >3.5)with positive Amsel's test.

Fig. 3
Fig. 3 Association between pseudo-time labels and BV-related clinical indicators.a Shannon-diversity index as a function of pseudo-time label in healthy arms.Each plot represents a different CST arm and colors indicate subCST, including in total 4627 healthy samples from all five datasets.The black line represents a linear regression to transformed polynomial values of pseudo-time.The R 2 and p value presented in each panel were determined by Spearman correlation test.b Receiver operating characteristic (ROC) curves for predicting six BV indicators with pseudo-time labels.The area under the ROC curve (AUC) is presented at the bottom of each plot.Colored curves represent prediction obtained using shuffled indicator labels.Bottom plots represent similar analysis, based only on held-out samples.The manifold-based curves are constructed using a range of 677-4055 samples (depending on the datasets that included the required data), while the held-out-based curves are generated using between 251-501 samples. ).

Table 2 .
Details of the five datasets included in the analysis.